Other cell culture assays such as 3--2, 5-diphenyltetrazolium bromide (MTT) and reactive oxygen species (ROS) monitoring were used to see the effect of inducers on the cellular system. MTT assay showed cell toxicity of MTX-NLPHS reduced at the lower concentration of the MTX, however, toxicity increased at the higher concentration of the MTX as compared to free MTX. ROS monitoring c revealed more scavenging of ROS using MTX-NLPHS as compared to free MTX. Confocal microscopy suggested the MTX-NLPHS induced more nuclear elongation with cell shrinkage comparatively.The focus of this research is to design a bioengineered drug delivery vehicle that is efficient in anti-cancer drug delivery in a controlled manner. The experimental work focuses on constructing a methotrexate-loaded nano lipid polymer system (MTX-NLPHS) that can transport methotrexate (MTX) in MCF-7 cell lines in a controlled manner through endocytosis via phosphatidylcholine. In this experiment, MTX is embedded with polylactic-co-glycolic acid (PLGA) in phosphatidylcholine, which acts as a liposomal framework for regulated drug delivery. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and dynamic light scattering (DLS) were utilized to characterize the developed nanohybrid system. The particle size and encapsulation efficiency of the MTX-NLPHS were found to be 198 ± 8.44 nm and 86.48 ± 0.31 %, respectively, which is suitable for biological applications. The polydispersity index (PDI) and zeta potential of the final system were found to be 0.134 ± 0.048 and -28 ± 3.50 mV, respectively. The lower value of PDI showed the homogenous nature of the particle size, whereas higher negative zeta potential prevented the system from agglomeration. An  Nanocarrier, a colloidal system, has been widely investigated in therapeutic directions that is smaller than 500 nm in size Liposomal drugs have some disadvantages related to chemical and physical stability, sterilization, batch-to-batch reproducibility, manufacturing scale-up as well as drug entrapment Phosphatidylcholine , PLGA , MTX , 2',7'-dichlorofluorescein diacetate dye , 4',6-diamidino-2-phenylindole , and propidium iodide  were obtained from Sigma Aldrich, USA. MCF-7 (human breast cancer cell line) was bought from National Centre for Cell Sciences (NCCS), Pune, India. Moreover, trypsin-ethylenediamine tetra acetic acid , chloroform , dimethyl sulfoxide  were purchased from Merck Millipore, USA. The remaining solvents and reagents were either analytical- or HPLC-grade purchased from SRL, India.The MTX-NLS and MTX-NLPHS were synthesized using a single emulsion solvent evaporation method The diameter, zeta potential, polydispersity index (PDI), of MTX-NLS and MTX-NLPHS in an aqueous solution was determined by DLS . In a brief, each sample was diluted fifteen times with organic solvent. The diluted solution (100 μg/mL) was then kept in a sonicator for 2 min at 30% amplitude. The cold condition was maintained throughout the sonication. The sonicated sample was collected in polystyrene cuvette at room temperature, and the particle size was measured at a constant 90° angle.To investigate the morphological characteristics of MTX-NLS and MTX-NLPHS, SEM (Evo-Scanning Electron microscope MA15/18) was utilized. The reference voltage on the SEM was 26 kV. Briefly, 1 mg of lyophilized sample was dissolved in 5 mL of solvent. Subsequently, sonication was allowed under cold conditions to separate the particles. 12 μL of the sample was placed on a rectangular coverslip and separately dried under reduced pressure. The sample was coated with gold to enable conductivity. Before the completion of the scanning, the conductive sample was held on a copper stub.2, PO43-, NH2, NH-CH2)) intact with them. A hydraulic press  producing 15 tons of force was used to make 13 mm pellets. The dried samples (1% by weight) of MTX-NLS and MTX-NLPHS were mixed individually with the potassium bromide (KBr) while making the pellets. Following that, the obtained pellets were scanned and data was captured from wavenumber 4000 cm-1 to 500 cm-1.The IR spectra of MTX-NLS and MTX-NLPHS were analyzed using IR  to validate the various functionalities   supplemented with 10% fetal bovine serum (FBS) , 100 U/mL penicillin/streptomycin . The cells were maintained in a humidified incubator at 37°C in an atmosphere of 5% CO2.MCF-7 cells were seeded in triplicate in a 96-well plate at a density of 10000 cells/well. Following overnight adherence, the media was replaced with a fresh medium containing treatments comprising MTX-NLS and MTX-NLPHS in five independent groups to see their comparative effects on MCF-7 cells. In each group, MTX concentrations varies from 20 μg/mL to 100 μg/mL. For all of the tests, the treated cells were incubated for 48 h. After incubation, media was decanted and the cells were then rinsed thrice with nuclease free water to discrete the remaining amounts of formulations. The cells were subsequently incubated with 20 μL of freshly prepared MTT (5 mg/mL) in PBS at 37°C for 4 h. After 4 h, the supernatant was removed and the formed formazan crystals were then dissolved using 100 μL of DMSO, and the cell viability was determined by measuring absorbance at 595 nm using an ELISA plate reader.4 cells. Further, cells were induced with 50 μM H2O2 solution (as a positive control), free MTX, and MTX-NLPHS for 48 h, however, cells without treatment were taken as a negative control. The experiments were performed in triplicate. After 48 h of incubation, cells were washed with 1X PBS and further incubated with 10 μM of DCFDA dye for 1 h and further fixed using 4% paraformaldehyde on the glass slides to observe changes with confocal microscopy. The interaction of DCFDA dye with intracellular ROS produces green fluorescence, which can be measured at excitation/emission spectra of 485/535 nm.This investigation was conducted for real-time monitoring of ROS in MCF-7 cells using cell permeable ROS sensor dye 2',7'-dichlorofluorescein diacetate (DCFDA). After this, cells were taken in a 6-well plate with each well having 1×104 cells per well in a 6-well plate and incubated overnight for cell adherence. Further, cells were treated with MTX and MTX-NLPHS and incubated for 48 h. Further, the treated cells were rinsed with PBS and then fixed with 4% paraformaldehyde for 15 min at 4 °C. The cells were again rinsed with PBS and then incubated with 1 μg/mL DAPI and 2 μg/mL PI in the designated wells for 30 min and 5 min respectively in the dark at RT. After incubation, cells were rinsed with PBS and then visualized under confocal microscope using excitation/emission spectra of 358/461 nm and 535/617 nm for DAPI and PI respectively. The images were taken at 20X magnification.To analyze the morphology of MCF-7 cell lines, DAPI and PI dyes were used. DAPI is a blue-fluorescent producing nuclear staining dye that binds to the minor groove of DNA by permeating the cell membrane whereas PI is a red-fluorescent producing nuclear dye that can only permeate dead cells Table . The relatively higher encapsulation efficiency in percentage is found due to the PLGA matrix in the core of the hybrid system. Results can be interpreted that when the diameter of the carrier grows, the effectiveness of the drug encapsulation increases. A nanoparticle analyzer was used to determine the average diameter of MTX-NLS and MTX-NLPHS. The particle size distribution of MTX-NLS and MTX-NLPHS was found to be in the range of 100-200 nm Table  Fig. 2)2). The relatively larger size of the MTX-NLPHS is observed due to the blending of PLGA with lipid molecules. An essential parameter that provides distribution of particle size is the PDI. The PDI value of MTX-NLS and MTX-NLPHS was found to be less than 0.2, which means the particle size distribution is likely to be uniform. The above findings are also comparable with previous studies, where particle sizes were distributed equally Table 1. The overall surface charge is obtained because of the phosphate group of the lipid. The reduction of the negative charge on the surface of MTX-NLPHS as compared to MTX-NLS is due to the phosphate group may interact with PLGA molecules through various interactions in the system. Furthermore, the sizes reported by the zeta sizer differ from the sizes obtained by SEM. The zeta sizer examines average particle size while the SEM governs individual particle size, which could account for the size discrepancy. Hydrodynamic diameters of the system obtained are in the nanoscale range, making it appropriate for a wide range of biological applications.The encapsulation efficiency of the formulated system is one of the relevant criteria to determine its application. The percentage of drugs entrapped in MTX-NLS and MTX-NLPHS was found to be 79.40 ± 0.28% and 86.48 ± 0.31%, respectively Fig. A, however, the SEM image of MTX-NLPHS showed a spherical shape with a relatively bigger size Fig. B. According to the aforementioned findings, the polymer's long chain may organize into its compact shape. As a result, the lipid monomer surrounding the polymer is consistently aligned and has the right shape with a relatively bigger size. This also means that the PLGA was successfully blended with the lipid system.SEM is used to see the surface topography of MTX-NLS and MTX-NLPHS. The spherical structure was seen in the case of MTX-NLS, showing the successful nanocarrier formulation using a single emulsion solvent evaporation method. The spherical structures of MTX-NLS were smooth and had a diameter of approximately 2 μm Infrared spectroscopy analysis was conducted to check the characteristics peaks of functionalities of MTX, PC, and PLGA and also used to validate the lipid-PLGA interactions in the final system.Fig. . Numerous peaks at wave numbers 3430.71 cm-1, 2917.70 cm-1, 2849.79 cm-1, 1647.2 cm-1, 1472.6 cm-1, 1233.25 cm-1, and 1064.99 cm-1, which correspond to OH, CH2, NH (stretching), NH (Bending), CH, CN, and PO4-3, are shown in the red spectrum (MTX-NLS). These peaks were seen because the formulated system contained MTX and lipids. The spectra in the blue curve (MTX-NLPHS) maintain all recognizable peaks with the shifted band. Interestingly, in this spectral region, a new peak was observed at a wavenumber of 1727.42 cm-1 which corresponds to the CO group. This signature peak is attributed to the presence of PLGA in the hybrid system. The unique peaks shifted to a higher wavenumber due to non-covalent interactions between PLGA and phosphatidylcholine. These comparisons show that the MTX-NLPHS was successfully formed.The FTIR spectra of MTX-NLS (red curve) and MTX-NLPHS (blue curve) showed numerous peaks in accordance with various functionalities Fig. . The diffractogram of MTX-NLS showed a characteristic peak at 2θ = 8.99°, which was attributed due to the presence of MTX. In the blue spectral region, a broader peak was observed which was due to the amorphous structure of the lipid. The diffraction peaks of the amorphous structure were scattered due to the absence of absolute positive and destructive X-ray interference in a finite-sized lattice. Interestingly, in the red spectra (MTX-NLPHS), the two characteristic peaks were observed which correspond to 8.97° and 9.31°. The new peak in the XRD pattern of the final system suggests that the lipid bilayer organizes and shifts the structure to the more crystalline structure in the presence of PLGA. In this system, the monomer PLGA may form the long chain of polymer inside the lipid nanocarrier. The characterization above clearly indicates that the successful formation of the MTX-NLPHS can be employed for further applications.The MTX-NLS and MTX-NLPHS were characterized using XRD to see the crystallinity pattern of the final system after incorporation of the PLGA Fig. 6, the drug dissociation pattern from the formulated nanohybrid system was examined for 250 h. Drug release from the constructed nanohybrid system was explained using zero-order and first-order kinetic models. The kinetic pattern depicted that the 18% drug was released within 20 h. The early fast rate of drug release could be attributed to some drugs being attached to the surface of the lipid. Furthermore, 200 h were required to release 71% of the drugs, and the cumulative percent of drug release (CDR) reached 90% in the 240 h that followed. The slow rate of drug release may ascribe to the presence of PLGA in the core of lipids. This system is helpful in suppressing the growth of cancer cells in a short time. In addition, a restricted or sustained rate of drug release from the constructed hybrid system may favor prolonged circulation of drugs in the blood circulatory system and also overcome the problem of multiple drug resistance in diverse biological applications. Therefore, our system met the requirement of a restricted and sustained rate of drug release after 20 h of incubation at the physiological pH. The constructive drug release pattern between zero and the first-order kinetic pattern was examined using a linear regression equation. Effective CDR is presented as the regression equation for the zero-order curve i.e., CDR= 17.57 ± (2.27) + -0.308 ± (0.01) [Time], with a R2 of 0.921. However, the effective CDR for the first order curve is represented as follows: 1.997 ± (0.021) + -0.0035 ± (0.0001) [Time] with an R2 of 0.930. The relatively higher R2 value for the first order model revealed that the hybrid nanocarrier released the drugs in a concentration-dependent phenomenon.We subsequently studied the drug dissociation in PBS after characterizing the nanohybrid system and studying the drug encapsulation. As shown in Fig. 7). MTX was used as a control (red bar) to compare with the formulated sample. The viability of the cancer cells was found to be 79.33 ± 3.8 %, 56.33 ± 5.7 %, 49.67 ± 4.6 %, 35 ± 5.9 %, and 22.33 ± 2.8 % for MTX at 20, 40, 60, 80, and 100 µg /mL, respectively. In another setup, the effect of MTX-NLPHS was examined on the cancer cells (black bars), where the percent viability was determined to be 90 ± 4.8 %, 60 ± 1.9 %, 39 ± 6.1 %, 15 ± 5.8 %, and 11 ± 2.7 % at 20, 40, 60, 80, and 100 µg /mL, respectively. The result suggested that the MTX reduced the cell viability following an increasing concentration of drugs. In this case, cells may uptake the drugs through passive diffusion and subsequently enhance cell toxicity. However, at the lower concentrations of MTX, MTX-NLPHS demonstrated considerably lower cell toxicity, although the toxicity increased with higher concentrations of MTX in the system. The result indicated that at lower concentrations of MTX in the system, the incorporation of PLGA may enhance its stability and control the drug release into the medium which thereby culminate to increase in the viability of the cells. Upon increasing the concentration of MTX in the system, the drug might be released from the carrier thus, disrupting its structural integrity and increasing cell death. The aforesaid findings suggested that the PLGA is a better carrier for the accumulation of MTX at optimum concentration within cells in a controlled manner. The IC50 value for the MTX-NLPHS system was found to be 51.78 µg /mL.To assess the cytotoxicity of MTX and MTX-NLPHS on cells, the MTT assay was utilized . Exposure of MCF-7 cells to 50 μM H2O2 solution significantly increased the formation of ROS which can be represented in terms of mean fluorescent intensity which was found to be 12 a.u.  compared to untreated cells for which mean fluorescent intensity was found to be 1.39 a.u. . Treatment of cells with MTX significantly attenuated the ROS formation which can be indicated in terms of mean fluorescent intensity of 11 a.u. . Further, more attenuation in ROS formation can be seen in the cells exposed to MTX-NLPHS indicated by mean fluorescence intensity of 3.80 a.u.  which infers more accumulation of drugs inside the cells through hybrid nanocarriers. These results further potentiated the selection of PLGA as a better carrier for controlled transport of drugs into the cells by endocytosis.The fluorescent probe DCFDA was used to measure intracellular ROS levels in cells treated with MTX and MTX-NLPHS ( Fig. . This finding suggests that cell fusion followed by carrier-mediated endocytosis is presumably the mechanism behind the cellular basis for the internalization of MTX-NLPHS The morphological changes induced by MTX and MTX-NLPHS were analysed with DAPI and PI staining using confocal microscopy. DAPI and PI stained cells when exposed to MTX-NLPHS cause higher nuclear elongation, cell shrinkage, and disintegration as compared to MTX-treated and untreated cellsin vivo tests will be conducted to check the efficacy of the constructed MTX-NLPHS.In the experimental work, we have designed NLPHS for MTX drug delivery in MCF-7 cells. Phosphatidylcholine blended with PLGA works as a scaffolding material in which MTX is incorporated for controlled drug distribution. The hybrid system was characterized using SEM, FTIR, XRD, and DLS and showed uniform size with an irregular shape, various functionalities, crystallinity, and chemical stability. The release kinetics pattern of the MTX-NLPHS depicts the controlled delivery of MTX and the system may overcome the problem of multiple drug resistance. Cell culture-based tests such as MTT assay and ROS monitoring analysis confirm the effect of NLPHS on the cellular system. Further, confocal microscopy suggests that the cell morphology significantly changed after treatment with MTX-NLPHS, which again potentiates the use of NLPHS as a better carrier for controlled drug delivery into the cells. Moreover,


Doxorubicin (DOXO) is a potent chemotherapeutic drug widely used to treat various cancers. However, its clinical application is limited due to serious adverse effects on dose-dependent cardiotoxicity. Although the underlying mechanism has not been fully clarified, DOXO-induced cardiotoxicity has been mainly attributed to the accumulation of reactive oxygen species (ROS) in cardiomyocytes. Fucoidan, as a kind of sulphated polysaccharide existing in numerous brown seaweed, has potent anti-oxidant, immune-regulatory, anti-tumor, anti-coagulate and anti-viral activities. Here, we explore the potential protective role and mechanism of fucoidan in DOXO-induced cardiotoxicity in mice. Our results show that oral fucoidan supplement exerts potent protective effects against DOXO-induced cardiotoxicity by reducing oxidative stress and preventing mitochondrial function injury. The improved effect of fucoidan on DOXO-induced cardiotoxicity was evaluated by echocardiography, cardiac myocytes size and cardiac fibrosis analysis, and the expression of genes related to cardiac dysfunction and remodeling. Fucoidan reduced the ROS content and the MDA levels but enhanced the activity of antioxidant enzymes GSH-PX and SOD in the mouse serum in a DOXO-induced cardiotoxicity model. In addition, fucoidan also increased the ATP production capacity and restored the levels of a mitochondrial respiratory chain complex in heart tissue. Collectively, this study highlights fucoidan as a potential polysaccharide for protecting against DOXO-induced cardiovascular diseases. Heart diseases and cancer are the main causes of mortality and morbidity in industrial countries, in particular, cancer remains the most common cause of death around the world ,2. ChemoFucoidan is a natural active sulfate polysaccharide mainly existing in the cytoplasmic matrix of brown algae , which hThe heart is the largest energy-consuming organ in the human body, and one of the cells with the most amount of mitochondria is cardiomyocytes. Almost 95% of ATP consumed by the heart comes from the oxidative metabolism of mitochondria . NotablyWe designed experiments to detect the protective effect of fucoidan on DOXO-induced heart damage in mice A. One grp < 0.001), that the LV end-diastolic internal dimension (LVIDd) is 3.882 ± 0.022 mm in the DOXO group vs. 3.197 ± 0.076 mm in the ctl group (p < 0.001), that the LV end-systole volume (LVESV) is 16.279 ± 0.660 mm3 in the DOXO group vs. 9.177 ± 0.817 mm3 in the ctl group (p < 0.001) and that the LV end-diastolic volume (LVEDV) is 63.031 ± 1.184 mm3 in the DOXO group vs. 41.128 ± 2.323 mm3 in the ctl group (p < 0.001)  is 2.456 ± 0.081 mm in the DOXO group vs. 1.432 ± 0.043 mm in the ctl group (< 0.001) . Therefo.996 mm3 , all of Two important markers of DOXO-induced cardiotoxicity, cardiomyocyte size and cardiac fibrosis, were analyzed by H&E staining and PicroSirius red staining. The results of H&E staining showed that myocardial cells in the DOXO group were smaller and that interstitial cracks were larger than those in the ctl group, while these lesions were significantly ameliorated in mice given fucoidan gavage A. In addANP), brain natriuretic peptide (BNP) and Myosin Heavy Chain 7 (Myh7) increased in the mice of the DOXO group, but these genes expression decreased in the mice pre-treated with fucoidan compared with those in the DOXO group (CTGF) and matrix metalloproteinase-2 (MMP-2) significantly increased after DOXO injection, which was consistent with the increases in ANP and BNP, and the combination treatment with fucoidan can alleviate these increases  and mitochondrial ATPase6 (mt-ATP6) -related mRNA expression of mitochondrial respiratory chain complex І–V in the heart tissues of each mouse  B. The mRch mouse C. The ex2, but it was increasing exponentially at higher doses  = , aspartate aminotransferase (AST) and lactate dehydrogenase (LDH-L) were measured by their respective kits . Creatine kinase isoenzyme (CK-MB) and serum lactate dehydrogenase isoenzyme 1 (LDH-1) were measured using their respective kits . Superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-PX) were measured by their respective kits . All of the mentioned indicators were tested according to the manufacturer’s instructions.Hearts from each mouse were excised and washed in cold phosphate-buffered saline , then fixed overnight in 4% paraformaldehyde (pH 7.4) and embedded in paraffin. Cardiac fibrosis was measured by staining 5 μm-thick tissue sections serially, and the slices were dewaxed, rehydrated and subjected to hematoxylin-eosin (H&E) for histological examinations with an optical microscope. The cross-sectional cardio-myocyte area was analyzed by Image J software .The sections were stained with the Picro Sirius Red Stain Kit  to evaluate interstitial fibrosis. Heart slices were dewaxed, rehydrated and incubated with the Weigert ferrolin staining solution for 10–20 min, washed with distilled water, incubated with Picro Sirius Red for 1 h, then washed with water, dehydrated and mounted. ImageJ software was used to measure the positive staining (red) fibrotic area with the average percentage of fibrosis to the total area, which was calculated for six images of each heart.Gapdh. Specificity was verified by the melting curve while each reaction was performed in triplicate, and the differences in gene expressions were calculated using the 2−ΔΔCt method.The total RNA of the mice heart was separated and extracted using TRIzon Reagent ; then, the Nanodrop 2000  was taken to measure the purity and concentration of the RNA. Using the HiScript III RT Super Mix  for qPCR reverse transcription to obtain the cDNA; then, the expression quantities were measured using Taq Pro Universal SYBR qPCR Master Mix . All of detective procedures followed the manufacturer’s instructions. Quantification was performed on the detective system and presented relative to The enhanced ATP Assay Kit  was used to detect ATP content in mice cardiac tissues. At first, the ATP working solution was added to the test wells to remove the background and then mixed with the tissue supernatant obtained from lysis for more than 2 s; finally, the RLU value was measured by a spectrophotometer. The efficiency of the test method can be referenced from the manual. The ROS content in the mice heart was evaluated using a fluorescent probe DCH-DA kit , and we conducted the experiments according to the manufacturer’s instructions. The heart tissue was lysed and added to DCFH-DA, incubated for 20 min in a constant temperature incubator at 37 °C and then cleaned with 1% sterile PBS three times. The obtained solution was added to a fluorescent microporous plate for quantitative analysis.t test, and one-way analysis of variance (ANOVA) with Bonferroni correction was used to compare three or more conditions. Statistical significance was considered when the probability values  were less than 0.05.Data in this study were presented as mean ± standard error of mean (SEM), and all statistical calculations were analyzed according GraphPad Prism 8 software . The difference between two conditions were compared using an unpaired Student’s


Young's modulus of 3D-printed PEEK suffered a reduction of 30% in relation to bulk PEEK. Customized 3D-printed PEEK endoprostheses had promising outcomes for the tibiae of 20 kg dogs. Pure compression tests of the non-inserted endoprostheses showed a maximum force of 936 ± 199 N. In the bending tests of non-inserted endoprostheses, the PEEK part remained intact. Quasistatic mechanical tests of bone-inserted endoprostheses (compression-bending and pure compression tests) reached a maximum force of 785 ± 101 N and 1,642 ± 447 N, respectively. In fatigue tests, the samples reached 500,000 cycles without failure or detriment to their quasistatic results. These outcomes surpass the natural weight-bearing of dogs, even during a galloping pace. In conclusion, the 3D-printed PEEK part of the endoprosthesis for an exo-endoprosthesis can withstand loading, even during a galloping pace.Exo-endoprosthesis is a limb salvage procedure for animals, although only expensive metal devices have been described. Now-a-days, new materials for this type of implant could be considered due to novel and affordable manufacturing techniques. However, a factor of safety (FoS) should be considered. There are kinetic and kinematic studies of canine natural gaits, which can be used to establish an FoS for mechanical tests for new non-metallic devices. Polyetheretherketone (PEEK) is used in different specialties in human medicine. Its mechanical properties  make this polymer an alternative to metals in veterinary traumatology. PEEK could also be used in 3D printing. The suitability of a novel inner part of an exo-endoprosthesis manufactured by fuse deposition modeling (FDM) was presented in this study for long canine bones. Mechanical characterization of 3D-printed PEEK material and  Exo-endoprosthesis is a limb salvage procedure in humans and, to a lesser extent, in animals –5. BecauSeeking price reduction and avoiding the aforementioned complications related to metal stiffness, other materials could be considered for implants. The stiffness is directly related to the elastic properties of a material, such as Young's modulus, tensile strength, and bending strength among others . MateriaPolyetheretherketone (PEEK) is one of those alternative non-metallic materials for medical devices. PEEK is a thermoplastic of the polyaryletherketone family that is generally used for different specialties in human medicine, such as neurosurgery, traumatology, and dental and craniomaxillofacial surgery , 12. PEENow-a-days, thanks to additive manufacturing (AM), quite a range of materials can be considered for manufacturing small productions of final devices with a minimum cost , 16. Theex vivo mechanical testing of new load-bearing implants. Currently, published quasistatic and dynamic mechanical tests of other veterinary implants  should be determined to endorse the feasibility of the material for that activity and situation. Currently, the FoS and working load have not been determined for veterinary limb salvage devices. Working load is the applied load on the device during the activity. FoS is a ratio defined for any mechanical device to avoid failure during its work, expressed as the ratio of the maximum load (failure load) to the working load. As a working load, these devices will withstand different body weights (BWs) according to the type of pace and moment of the stance phase –27 durinimplants , 32, 33 implants , while aimplants . These dimplants . Stridesex vivo research on the use of a PEEK sleeve to interface between the bone and inner part of an exo-endoprosthesis in the context of limb-sparing surgery. The 3D-printed endoprosthetic element was tested by new loading protocols to mimic normal canine loads during walking, trotting, and galloping. To our knowledge, no evidence of mechanical tests was found for other exo-endoprostheses in veterinary medicine.The aim of this study is to determine if PEEK material in contact with the bone could be a possible weight-bearing option for canine patients during their regular gaits. This work describes initial The assessed endoprosthesis was patented by Mendaza et al.  and was The PEEK sleeve of the endoprosthesis was designed with a CAD program . The external diameter of the stem was 0.40 mm larger than the drilled medullary canal of a tibia (9 mm). Thus, all external diameters of the stems measured 9.40 mm. The diameter of the inner cavity of the PEEK sleeve was 6 mm just like the diameter of the threaded rod. This cavity was slightly reduced at the level of the stem for applying extra-radial compression to the bone, such as that described by Mendaza-DeCal et al. .Tibia endoprostheses and tensile and bending test samples were printed in PEEK . Filaments were stored in a special ziplock multi-layered bag with a special EVOH barrier film and silica desiccant sachets provided by the filament supplier. Otherwise, following the recommended protocols in the literature  and by fTensile and bending test samples and endoprostheses were printed by FunMat HT  based on FDM technology, with a build volume of 260 mm × 260 mm × 260 mm . The printer has a hardened steel nozzle of 0.4 mm diameter. This printer reaches a nozzle temperature of up to 450 °C, bed temperature of up to 160 °C, and chamber temperature of up to 90 °C. Before printing, 3D virtual models were sliced by Simplify 3D software .*50.21*14.72 mm3, which are x, y, and z of the printing bed, respectively. Also, the bending test samples were printed like rectangular bars, with the same printing orientation as the other 3D models. A horizontal orientation was carried out to take into account the main mechanical forces borne inside the medullary canal. The general printing parameters were constant for all 3D models, which are shown in All 3D models were horizontally oriented with respect to the printing bed . The larFurthermore, immediately before each printing, a specific liquid fixative  for 3D printing was applied to the cold print bed for better adhesion of the material during printing. A preheat of the chamber and build bed temperatures were set and allowed to stabilize for at least for 30 min before starting any printing.Ex vivo fresh tibiae were used to insert the endoprostheses. The 33 tibiae used for this study belonged to canine specimens (20.85 ± 1.25 kg BW) that were euthanized for reasons unrelated to this study. The longitudinal length of tibiae was 208.51 ± 40.27 mm. The fresh bones were immediately stored inside a vacuum bag in a freezer until the insertion of the endoprosthesis. Bones were perpendicularly cut to the longitudinal axes above the distal epiphysis. Immediately, the medullary canals of the tibiae were measured on the cut plane by a metric digital caliper. Tibiae with an average medullary canal diameter of 9.48 ± 0.29 mm were selected. This average medullary canal diameter was observed on the selected canine specimens. After measure, the medullary canal was drilled with a 9 mm bit to homogenize the canal. Two PLA 3D-printed self-designed surgical guides were used to make perpendicular cuts and to align drilling . In. InEx viOnce the endoprostheses were inserted, 25 tibiae were attached with a two-component epoxy resin  in aluminum holders. Tibiae were cured in the holders at half the average angle of the stifle joint when the dog is standing , as descThe tensile properties of the 3D-printed PEEK were determined using tensile test samples with dog bone shapes (DB-samples)  Figure . The dimA three-point bending load arrangement was used to determine the bending properties in accordance with ISO 178 . The tesThe results of the elastic properties were compared with those of canine tibiae, bulk PEEK, 316L stainless steel (316L-SS), and titanium (Ti) obtained from the literature .Pure compression  and bendin vivo conditions. Eight ET-samples were evaluated by pure compression testing  were prepared as described in section 2.4 above for pure compression (90°) and compression-bending test (67°). Each of the ET samples was assessed using the universal testing machine Elib 20 W  with a load cell of 2 kN . Tests w testing  and 15 E testing  at a croFatigue tests are particularly important to study the long-term behavior of the endoprosthesis-tibia construct. The load to be supported by the tibia can be approximated by equation 1 , 30. ForR = 0.1. A frequency of 2 Hz was used for the test, which is similar to the pacing frequency when the dog is trotting. Under these conditions, 10 ET-samples were evaluated.Fatigue testing was conducted under room conditions (22 ± 1 °C) using an electrodynamic testing machine with a load cell of 3 kN . ET-samples were prepared as described in section 2.4  for compp ≤ 0.05) by the F-test for both comparisons.Data from pure compression of E-samples and ET-samples tests were analyzed by analysis of variance (ANOVA) using the STATGRAPHICS program . Also, compression-bending data with or without fatigue tests were analyzed by analysis of variance (ANOVA). Significant differences were observed  was assessed for the 15 ET-samples . The tesThe results of the pure compression test reached a maximum force of 1,642 ± 447 N before failure . For higp ≤ 0.05. The calculated FoS was 2.5 ± 0.2 for ET-samples at the compression–bending test after fatigue.Upon fatigue test, the assessed ET-samples reached 500,000 cycles without failure. To analyze the effect of fatigue, the survivor samples were evaluated for failure by means of the quasistatic compression-bending test described in section 2.5.3. The samples could stand 764 ± 62 N upon static compression-bending test after fatigue before breaking . No statThe 3D-printed endoprosthetic element can withstand normal weight-bearing loads reported in dogs at walk and trot. Additionally, we demonstrate that the behavior of the construct with acute loading to failure is not significantly different if the endoprosthesis is pre-fatigued with 500,000 cycles of pre-loading. The PEEK part of the endoprosthesis bore higher flexion and compression forces than canine tibiae during galloping , 30. AllThe ET-samples showed a maximum load at failure of more than 2-fold the selected working load (300 N for a 20 kg dog) in the compression–bending test. These results are even more promising considering that we only used a wet canine tibia without the remaining skeletal system components of the pelvic limb. Besides, we are comparing compression-bending results with the force borne by the whole pelvic limb, which is another handicap for the studied endoprosthesis. In addition, it should be considered that the compression-bending tests were carried out in a standing-like position, imitating the canine stifle angle . It meanThe fatigue test shows the long-term behavior of a device. The results of fatigue and the following compression-bending test indicate that the endoprosthesis can withstand 500,000 cycles with a trotting-like frequency and the total BW during galloping without detriment of its strength. The fatigue parameters, cycles, and frequencies were set based on other papers where fatigue resistance was evaluated for different metal implants , 33 and Pure compression results confirm that force-bearing data from only pure compression of the E-samples or from pure compression of ET-samples are significantly different. The ET-samples could stand 1,642 ± 447 N on pure compression (5-fold increase in the dog's weight-bearing), in contrast to 936 ± 199 N in the E-samples. This means that the endoprosthesis has better results in an environment closer to a real one, such as using wet bones  as recepWhen a 3D printer is used to obtain solid pieces, the resultant material shows anisotropic behavior . TherefoIn conclusion, the evaluated endoprosthetic part of an exo-endoprosthesis can largely withstand a dog's weight during a galloping pace and at a higher frequency than this pace without detriment to its maximum weight-bearing. This makes the 3D-printed PEEK exo-endoprostheses a suitable mechanical choice for medical devices in veterinary medicine.The original contributions presented in the study are included in the article/Conceptualization: RM-D, YB, JR-R, and JR-Q. Data curation, methodology, resources, and writing—original draft: RM-D, YB, and JR-R. Formal analysis and validation: RM-D and JR-R. Funding acquisition and project administration: JR-Q and SP-F. Investigation: RM-D, YB, JR-R, and EP. Software: RM-D, SP-F, EP, YB, and JR-R. Supervision: JR-Q, SP-F, and JR-R. Writing—review and editing: RM-D, YB, JR-R, and JR-Q. All authors contributed to the article and approved the submitted version.This work was supported by the regional government of Madrid, Spain [Grant IND2017/BMD-7726]. RM-D received a PhD fellowship from this grant. The regional government of Madrid was not involved in any decision about the development of this study.RM-D and SP-F were employed by ABAX Innovation Technologies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Glycyrrhizae radix et rhizome (GRR) is commonly made into a charcoal processed product, termed GRR Carbonisatas (GRRC), for use in traditional Chinese medicine (TCM). GRRC is widely used to treat MPS and other gynecological diseases. In this study, GRRC was prepared through pyrolysis. Subsequently, GRR-derived carbon dots (GRR-CDs) were purified through dialysis and characterized using transmission electron microscopy, high-resolution transmission electron microscopy, Fourier-transform infrared, ultraviolet, fluorescence, X-ray photoelectron microscopy, and high-performance liquid chromatography. The effects of GRR-CDs on MPS were examined and confirmed using ovariectomized female mice models. The GRR-CDs ranged from 1.0 to 3.0 nm in diameter and with multiple surface chemical groups, as indicated by the results. GRR-CDs can elevate the estradiol (E2) level of healthy female mice. Moreover, GRR-CDs can alleviate MPS using the typical ovariectomized mice model, as confirmed by elevating the estradiol (E2) level and reducing the degree of follicle stimulating hormone (FSH) and luteinizing hormone (LH) and raising the degree of uterine atrophy. The results of this study suggested that GRR-CDs may be a potential clinical candidate for the treatment of MPS, which also provides a possibility for nanodrugs to treat hormonal diseases.With the extension of the human life span and the increasing pressure of women’s work and life, menopause syndrome (MPS) refers to a problem that puzzles almost all women worldwide. Hormone replacement treatment (HRT) can effectively mitigate the symptoms but can also exert adverse effects to a certain extent.  Menopause is defined retrospectively as 12 months of amenorrhea for no other explanations, i.e., the termination of female reproductive capacity; it has been often accompanied by a variety of health issues that may affect all women ,4,5. BesCarbon dots (CDs) have become a rising star in carbon nanomaterials with a size of smaller than 10 nm in diameter ,12,13). Glycyrrhizae radix et Rhizoma (GRR) is one of the most frequently used Chinese herbs which is called Gancao in Chinese. It has been documented since the Qin and Han Dynasties. GRR Carbonisatas (GRRC), the product of GRR after high temperature, was first documented in the Treatise on Febrile and Miscellaneous Diseases of the Eastern Han Dynasty and was extensively employed in many different diseases thus far . Our previous research has suggested that CDs generated by high-temperature heating are correlated with the actions of charcoal drugs. The therapeutic effect of GRR-derived carbon dots (GRR-CDs) on MPS was explored according to the TCM clinical application and our preliminary results.In this study, we performed replicate synthesis three times to ensure that GRR-CDs possessed reproducible properties. Then, GRR-CDs were identified and characterized. The experimental process is shown in The synthesis yield of GRRC was (38.52 ± 0.45)%. The synthesis yield of GRR-CDs was (0.17 ± 0.01)%.A transmission electron microscope (TEM) was used to describe the morphology and size of GRR-CDs A. Their The Fluorescence (FL) spectrum showed that the maximum excitation wavelength of GRR-CDs was 321 nm and that the maximum emission wavelength was 415 nm D. The qu−1 was assigned to the absorption bands of O-H and N-H stretching vibrations while C-H stretching vibrations appeared at 2923 cm−1. The measurement of 1631 cm−1 corresponds to C=O stretching. C-O-C bonds were identified at 1097 cm−1, and the peak at 1384 cm−1 was attributed to C-N stretching. The FTIR spectrum of GRRC without dialysis, GRRC powder without boiling, and GRR were also described (The characteristic peaks of purified GRR-CDs F were inescribed F. The tyThe ultraviolet-visible (UV-Vis) absorption spectrum G showed As depicted in the X-ray photoelectron spectroscopy (XPS) A, the thThe High-performance liquid chromatography (HPLC) results of GRR and GRR-CDs were shown in As depicted in Compared with the E2 level in the control group (145.37 ± 13.45 nmol/L), E2 levels significantly increased in the high (184.00 ± 12.45 nmol/L), medium (176.84 ± 17.05 nmol/L), and low (170.90 ± 10.84 nmol/L) doses of GRR-CDs groups.p < 0.05) compared with the control group (5.19 ± 0.49 nmol/L).The T levels of high- (5.21 ± 0.49 nmol/L), medium- (5.10 ± 0.38 nmol/L), and low-dose (5.34 ± 0.38 nmol/L) GRR-CDs groups showed no significant difference , follicle stimulating hormone (FSH), and luteinizing hormone (LH)) were investigated to investigate the therapeutic effect of GRR-CDs on climacteric syndrome.In addition, as depicted in As depicted in Estrogen, a sex hormone contained in both men and women, is especially one of the most important hormones in the female body of all ages and has a wide range of biological activities. There are three estrogens naturally produced in the female body: estradiol (E2), estrone (E1), and estriol (E3). E2, the main circulating estrogen, is in the largest quantity of these three estrogens and has the highest affinity for estrogen receptors . These sMPS has a high prevalence among ethnic groups, and it is correlated with the majority of women worldwide. It is a natural process that occurs in women’s lives when the ovaries have complete (or near-complete) follicular exhaustion, resulting in significantly elevated FSH and very low estradiol levels in serum . It is aHormone replacement therapy (HRT) by estrogen-only or with progesterone has been confirmed as the most effective way in mitigating menopausal syndrome. HRT is capable of effectively controlling vasomotor symptoms and genitourinary syndrome of menopause . RandomiCDs become a potential choice for biomedical applications for their characteristics  . Our teaTreatise on Febrile and Miscellaneous Diseases, it was commonly made into GRRC for use. GRRC has been extensively used to treat gastrointestinal ulcers, abdominal pain, cough, arthritis, and so on. Besides, GRRC also plays a role in the treatment of menopausal palpitation, hot flashes, and other gynecological diseases. From GRR to GRRC, carbon dots were generated in the high-temperature process. Thus, we used GRR as carbon source to exploit a novel nanomedicine with the effects of increasing estrogen.GRR refers to a type of Chinese herbal medicine used commonly in TCM prescriptions for over two thousand years. According to TCM theory, GRR is mainly effective for tonifying fatigue and debilitation, relieving cough and asthma, and reducing drug toxicity. As one of the most frequently used traditional Chinese herbs in In this work, GRR-CDs were prepared by a simple and green calcination method at the condition of 375 °C for 1 h. As the only precursor for the synthesis of GRR-CDs, GRR was a type of Chinses medicine with the advantages of low cost, environmental affinity, and high yield, providing the possibility to produce GRR-CDs in a large scale. FTIR and XPS analysis has proved that active groups such as carboxyl and hydroxyl could be found on the surface of GRR-CDs, which may further provide an explanation for the prominent estrogen-increasing bioactivity of GRR-CDs. Compared with the FTIR spectra of GRR, GRRC without dialysis, and GRRC powder before boiling, the change in GRR-CDs surface group content may be the reason for its unique biological effects. HPLC was used to compare the different components between GRR-CDs and GRR, indicating that small molecule substances were removed from the original.Given that GRR-CDs can significantly elevate estrogen levels in healthy female mice, the effects of GRR-CDs in facilitating pathological estrogen reduction were explored. The complete resection of the bilateral ovaries served as an MPS mice model to simulate the state of MPS in the female body. This method showed several advantages . In this study, E2, LH, FSH levels, uterus index, and pathological section of the uterus were selected as the observation targets to explore the effect of GRR-CDs. The positive effects of GRR-CDs in elevating the E2 level has been demonstrated in the MPS model mice. Moreover, the elevated E2 level was indicated by the decreased FSH and LH levels. The above-described conclusion was further verified by the increase in the degree of uterine atrophy. As revealed by this result, GRR-CDs can supplement the E2 in women’s bodies while mitigating the symptoms correlated with MPS. Moreover, this indicates that GRR-CDs may also play a role in the treatment of other diseases related to lower estrogen or low estrogen status in different periods of women’s lives. However, the mechanism of GRR-CDs that elevated the E2 level has not been clarified in this study. The previous research by our team suggested that CDs may affect sex hormone levels by playing a certain role in lipid metabolism. In-depth research should be conducted to determine the underlying mechanisms and broader bioactivities.GRR originated from Beijing Qiancao Traditional Chinese Medicine Co., Ltd. , and GRR-CDs were prepared in our laboratory. Dialysis membranes  were purchased from Beijing Ruida Henghui Technology Development Co., Ltd. . Benzylpenicillin potassium for injection was offered by Jiangxi Keda Animal Pharmaceutical Co., Ltd. . Mouse E2, T, FSH, and LH Elisa kits were offered by Kete Biotechnology Co., Ltd. . All other commercial chemicals and reagents employed in this study were purchased from Sino-pharm Chemical Reagents Beijing  and were of analytical reagent grade. All experiments were performed with deionized water.The protocols of this study conformed to the Guide for the Care and Use of Laboratory Animals and gained approval from the Committee of Ethics of Animal Experimentation of the Beijing University of Traditional Chinese Medicine (with the ethic code of BUCM-2022032401-1050). Female adult Kunming mice (weighing 25.0 ± 2.0 g) were provided by the SiBeiFu  Biotechnology Co., Ltd. All animals were placed in a controlled environment with the ambient temperature of 24.0 ± 1.0 °C, relative humidity of 55–65%, under a 12 h light/dark cycle, and with free access to food and water based on adaptive feeding for 7 days.GRR was placed in crucibles and covered with aluminum foil to form a sealed place. Subsequently, the GRR was calcined at 375 °C for 1 h in a pre-heated muffle furnace . GRRC after heating was weighed to determine the synthesis yield (SY) as follows:The GRRC was crushed into a fine powder after the furnace cooled to ambient temperature. GRR-CDs were purified by using the dialysis method. A total of 50 g powder was removed, mixed with 1500 mL deionized water (DW), and boiled at 100 °C three times in a water bath for one hour each time. The residue was removed by filtration through a 0.22 μm filter membrane, and the obtained solution was dialyzed against DW for 7 days using a dialysis membrane with a molecular weight cut-off of 1000 Da to obtain the purified GRR-CDs and then stored at 4 °C for further characterization and use. The GRR solution was prepared by boiling at the same condition and without dialysis. The weight of GRR-CDs was weighed after lyophilization. The SY of GRR-CDs was calculated as follows:The morphology and size of GRR-CDs were characterized using a transmission electron microscope  with an acceleration voltage of 200 kV. The atomic lattice fringes of the GRR-CDs were examined using high-resolution TEM . GRR-CDs were performed in KBr pellet, and the organic functional groups were recorded on a Fourier transform infrared spectroscopy spectrophotometer . The UV-Vis spectrum and fluorescence properties were acquired using UV-Vis  and fluorescence spectroscopy , respectively. The surface chemical groups and elemental composition information were analyzed by X-ray photoelectron spectroscopy . Q represents the QY; I denotes the integral area of emission intensity; “A” expresses the absorbance at 321 nm; “RA and CDsA were kept below 0.05 to minimize the effect of reabsorption.A quantity of 0.1 M quinine sulphate  served as a standard solution to determine the FQY of GRR-CDs, and was calculated as follows: High-performance liquid chromatography (HPLC) was used to evaluate the individual components before and after pyrolysis and dialysis to compare the difference between GRR and GRR-CDs. The fingerprint analysis was conducted using a ZORBAX-C18 column  based on an Agilent series 1260 HPLC instrument  equipped with a quaternary pump, a diode-array detector, a degasser, an autosampler, as well as a column compartment. The methanol extract of GRR and GRR-CDs was prepared and then processed under the same detection conditions. The mobile phase A (acetonitrile) and B (4% phosphoric acid) were filtered through 0.22 µm cellulose acetate membrane filters  prior to use. The gradient program is elucidated in the following: 0–15 min, 10–20% A and 90–80% B; 15–30 min, 20% A and 80% B; 30–40 min, 20–30% A and 80–70% B; 40–50 min, 30–10% A and 70–90% B. Furthermore, the column was held at 35 °C; the flow rate was set to 1.0 mL/min; and the detection wavelength was 254 nm.n = 24) were randomly assigned to four groups of six animals each as follows: control, high- (H), medium- (M), and low-dose (L) GRR-CDs treatment groups. Mice in the control group were administrated with NS . The high- (H), medium- (M), and low-dose (L) GRR-CDs treatment groups were administered 1.6 mg/kg, 0.8 mg/kg, and 0.4 mg/kg of GRR-CDs, respectively. Drugs were given by intragastric administration once a day continuously for 14 days.Female KM mice (n = 36) were randomly assigned to six groups of six animals each as follows: sham operation (Sham), MPS model (Model), positive drug (P), and high- (H), medium- (M), and low-dose (L) GRR-CDs treatment groups. Bilateral ovaries of mice were removed to build the MPS model except for the sham operation group. Mice that only removed some adipose tissue by the side of the ovary were employed as the sham group. Then the mice were applied the penicillin to the suture after the surgery and identified for 5 days. The sham operation and MPS model groups were treated by NS, and the positive drug group was administrated with estradiol valerate tablets . The high- (H), medium- (M), and low-dose (L) GRR-CDs treatment groups were administered 1.6 mg/kg, 0.8 mg/kg, and 0.4 mg/kg of GRR-CDs, respectively. Drugs were given by gavage administration once a day, and the treatment duration was 28 days.Female KM mice  as follows:After the treatment, blood samples were acquired by removing the eyeballs of mice. Next, the samples were centrifugated at 3000 rpm for 10 min to obtain the serum. E2 and T levels in healthy female mice and E2, FSH, and LH levels in the MPS mice were examined using Elisa kits  in accordance with the manufacturer’s instructions, respectively.The right side of uterus tissue samples were fixed in 10% neutral-buffered formalin at 4 °C for more than 48 h, dehydrated, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (H&E) for histopathological examination. Morphological changes were compared among the sham operation, model, positive drug, and GRR-CDs-treated groups.p value < 0.05 indicated statistical differences and p value < 0.01 indicated statistically significant differences.All data analyses were performed using IBM SPSS statistics software (version 26). Results were shown as means ± standard deviation (SD). Differences between groups were performed by one-way ANOVA followed by the least significant difference test. In this study, we successfully synthesized GRR into GRR-CDs by one-step pyrolysis. To the best of our knowledge, for the first time, this study proves that GRR-CDs could elevate the E2 level, decrease FSH and LH levels in serum, and moreover reduce the degree of uterine atrophy. This not only suggests the potential of GRR-CDs as a drug for alleviating menopause syndrome and its related symptoms, but also provides a potential possibility for nanodrugs to treat hormonal diseases.


Hyperlipidemia is traditionally considered a risk factor for diabetes. The effect of low-density lipoprotein cholesterol (LDL-C) is counterintuitive to diabetes. We sought to investigate the relationship between LDL-C and diabetes for better lipid management.We tested the shape of association between LDL-C and diabetes and created polygenic risk scores of LDL-C and generated linear Mendelian randomization (MR) estimates for the effect of LDL-C and diabetes. We evaluated for nonlinearity in the observational and genetic relationship between LDL-C and diabetes.P=0.0168) in an observational analysis which was more prominent in MR analysis and suggested increasing the overall distribution of LDL-C in females led to an overall decrease in the risk of diabetes (P=0.0258).Traditional observational analysis suggested a complex non-linear association between LDL-C and diabetes while nonlinear MR analyses found no evidence for a non-linear association. Under the assumption of linear association, we found a consistently protective effect of LDL-C against diabetes among the females without lipid-lowering drugs use. The ORs were 0.84 (95% CI, 0.72–0.97, We verified the liner protective effect of LDL-C against diabetes among the females without lipid-lowering drug use. Non-linear associations between LDL-C against diabetes in observational analysis are not causal. Diabetes is a complex metabolic disease regulated by a combination of environmental and genetic factors. Individuals with metabolic syndrome (MetS) have significantly increased risk for diabetes, independent of many other risk factors . MetabolAlthough the cluster of lipid abnormalities associated with type 2 diabetes is defined by a high concentration of TG and small dense LDL and a low concentration of HDL cholesterol , 5, the P<0.001; I2 = 0%)  . It’s stI2 = 0%) , 17. HowAnother reason for the controversial association of LDL-C with diabetes is the impracticability of excluding confounding factors and reverse causality from the observational study. Mendelian randomization (MR) analysis has the advantage to circumvent this problem to some extent, which uses germline genetic variants as an instrumental variable (IV) to proxy for environmentally modifiable exposures within observational epidemiological studies and is considered analogous to RCT due to the law of independent assortment .Therefore, in this study, we investigated both liner and non-liner associations between LDL-C and diabetes in observational and MR studies respectively to provide scientific basis for lipid management of diabetic patients.The present study relied on the Fangshan Family-based Ischemic Stroke Study in China (FISSIC) . FISSIC This study was approved by the Ethics Committee of the Peking University Health Science Center , and written informed consent was provided by all participants.In the FISSIC study, baseline data including sociodemographic status, education, occupation, diet, lifestyle, health behavior, and medical history, of all participants were collected through a face-to-face questionnaire survey by trained staff. For smoking, we assigned current smokers and former smokers as ‘ever smokers’ to avoid a misleadingly elevated risk for the reference group. Body mass index (BMI) was calculated as kg/m2. Seated position BP was measured after resting for at least 5 min. The systolic BP (SBP) and diastolic BP (DBP) used in the analysis were calculated as the mean of three consecutive measurements for each participant. Hypertension was defined as self-reported history of hypertension or SBP≥140 mmHg or DBP≥90 mmHg or use of antihypertensive medications. After overnight fasting of at least 12 hours, a venous blood sample was obtained from the forearm of each participant. Serum or plasma samples were separated within 30 minutes of collection and were stored at -80 °C, which were used for measurement of fasting blood glucose and the standard 75-g oral glucose tolerance test, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, creatinine concentrations, and DNA analysis. Laboratory tests of serum lipid levels, including TG, TC, HDL-C, and LDL-C, were performed by qualified technicians from the Laboratory of Molecular Epidemiology in the Department of Epidemiology at Peking University. Serum glucose, blood urea nitrogen, creatinine, concentrations of total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were measured by using the Hitachi 7180 autoanalyzer  . IndividOur primary outcome of interest was diabetes, which was defined if one of the following inclusions was met: 1) self-reported diabetes status; 2) hypoglycemic drugs use; 3) fasting blood glucose (FBG)≥7.0 mmol/L; 4) two hours blood after glucose oral glucose tolerance test (OGTT) ≥ 11.1 mmol/L. We took FBG as the secondary outcome to further assess the effect of LDL-C on diabetes. Individuals with missing values of the above variables were excluded.−4P<1 × 10). The correlation between allele frequency of our samples and those of East Asian samples from the 1000 Genomes Project was examined and a high correlation (2 =r0.98) was observed.DNA was extracted using a LabTurbo 496-Standard System . In addition, the purity and concentration of DNA were measured using ultraviolet spectrophotometry. Furthermore, the genomic DNA sample was genotyped with Infinium Asian Screening Array . We used two negatives (blanks) and three positive controls to control the quality of the genotyping process, and the results were satisfied. We also chose 5% samples randomly for repeat analysis to verify the reproducibility of the genotyping data. Plate-, individual-, and variant-level checks were conducted to exclude poor-quality genotype calls from the data set. The individual-based quality control criteria included a call rate of<99%, gender mismatch, excess heterozygosity, and relatedness. Variant-level quality control was performed to exclude variants with low cluster scores, low call rate (<99.9%), and those that deviated from Hardy–Weinberg equilibrium , and blood pressure. For linear regression of FBG, we added the additional adjustment for hypoglycemic drug use.We used restricted cubic splines (RCS)  to fit aWe generated the genetic risk score (GRS) of LDL-C to use as an instrumental variable in our MR analysis. The risk score for LDL-C in each of our genotyped participants is a weighted sum of the effect size estimates from the East Asian meta-analyses  and the -8P< 5×10) for association with LDL-C as candidate SNPs. Secondly, the SNPs were eliminated if they were in linkage disequilibrium (LD) based clumping at an LD threshold of 2r<0.001 for all variants within a 1Mb window and a secondary clumping with a threshold 2r<0.1 for all variants within the same chromosome using reference LD from the 1000 Genomes project. And the SNPs that were not available in our chip were not used in the analysis. Combined with the information of selected SNPs and weights, we specified the “sum” model in the software PRSice 2.3.5  to complete the calculation of GRS (https://shiny.cnsgenomics.com/mRnd/)  summary statistics for the genetics proxies of LDL-C of East Asian ancestry. Strict selection criteria were used to select qualified SNPs and construct GRS. We extracted only SNPs that pass the genome-wide significance threshold (n of GRS . Additiom/mRnd/) . The equm/mRnd/) .via linear regression to generate an estimate of the effect of the GRS on the LDL-C, adjusting for sex, age and age2. We use the F statistic of regression at this stage to evaluate the validity of GRS. F statistic >10 was considered valid. The associations of the resulting estimated LDL-C values were examined using linear regression with the same adjustments in an observational analysis.We performed a two-stage MR analysis to model the linear effect of LDL-C. In the first stage regression, we regressed LDL-C on the GRS nlmr R package .P<0.0001), obese (P<0.0001), former smokers (P=0.0004), have a diabetes family history (P<0.0001), have CHD (P<0.0001) and high blood pressure (P<0.0001), and use lipid-lowering drugs (P<0.0001). The levels of TG, HDL, and SBP were consistent with expectations. No significant difference was found in TC, LDL-C, and DBP.In aggregate, we included 4,876 individuals with sufficiently reliable baseline information and genotypes to evaluate the relationship between LDL-C with the risk of diabetes  and MR analysis (P=0.3648) with the poor power of 0.29 (P=0.0168) in observational study. This effect was more prominent under the causal inference framework in MR analysis and suggested that increasing the overall distribution of LDL-C in females per unit would lead to an overall decrease in the risk of diabetes by 56%  with the power of 0.91. We also found a similar protective effect of LDL-C against diabetes among the never smokers who did not receive the lipid-lowering drug both in observational analysis  and MR analysis . This was largely because the proportion of ever smokers was 64.2% among the males and 3.3% among the females (P=0.0234) , and blood pressure, we found no significant linear association between LDL-C and diabetes in all participants both in the observational analysis . The model revealed a significant U-shaped association (P for non-linearity<0.001). Below 3.2 mmol/L, the odds ratio per standard deviation higher predicted LDL-C for diabetes was 0.71 (0.58 to 0.87). Above 3.6 mmol/L, the odds ratio for genetically LDL-C for diabetes was 1.46 (1.14 to 1.86). This result persisted in subgroups without lipid-lowering drug use. Notably, among participants taking lipid-lowering drugs, there was no U-shaped association (P for non-linearity = 0.2144).We subsequently assessed for nonlinear associations of LDL-C with the risk of diabetes using restricted cubic splines in observational analysis. We finally identified three nodes and non-linear relationship between LDL-C and diabetes . Sensitivity analyses stratified by sex show no evidence of nonlinearity (P for nonlinearity >0.1). And it was worth noting that although the effects were not significant, there showed a heterogeneous trend in males and females  in all participants as well as participants without lipid-lowering drug use. The results were robust across genders and CHD-free population among the non-lipid-lowering participants. In MR analysis, per unit increase in LDL-C was causally associated with a 0.14 mmol/L decrease in FBG (P=0.0391) among participants who weren’t taking lipid-lowering drugs. The effect was also observed in females  and in CHD-free participants . We took a sensitive analysis by excluding participants with known diabetes or hypoglycemic drug use. The effects of LDL-C on FBG were attenuated and the significance was reduced. In observational analysis, per unit increase in LDL-C was associated with a 0.03 mmol/L increase in FBG (P=0.0161) in all participants as well as participants without lipid-lowering drug use. In MR analysis, there were no significant association between LDL-C and FBG except among the never smokers without lipid-lowering drug use.  , which makes the relationship between LDL-C and FBG more complicated. When LDL-C fell between 2.6 mmol/L and 3.8 mmol/L, per unit increase in LDL-C was associated with a 0.29 mmol/L increase in FBG (P=0.0069) in all participants and 0.40 mmol/L increase in FBG (P=0.0007) in participants without lipid-lowering drug use. Over 3.8 mmol/L, per unit increase in LDL-C was associated with a 1.01 mmol/L decrease in FBG (P=0.0139) in all participants and 1.38 mmol/L decrease in FBG (P=0.0015) in participants without lipid-lowering drug use. We observed no evidence of a nonlinear relationship in the MR analysis , but plots showed a sustained downward effect of LDL-C to FBG, though not significant (P=0.0056) in all participants and 0.43 mmol/L increase in FBG (P=0.0012) in participants without lipid-lowering drug use. Over 3.8 mmol/L, per unit increase in LDL-C was associated with a 1.21 mmol/L decrease in FBG (P=0.0090) in all participants and 1.49 mmol/L decrease in FBG (P=0.0019) in participants without lipid-lowering drug use. We still did not observe any evidence of a nonlinear relationship in the MR analysis  (KLF14 and ABCA8) showed female-specific association with TG and LDL-C, respectively (ESR α) gene  increased susceptibility to type 2 diabetes mellitus and may also impact serum lipid metabolism in Chinese Guangzhou women (P< 0.05) and positively correlated with LDL-C in men (P< 0.05)  . Richard = 0.03) . The tra = 0.03) . Teslovi = 0.03) . Two locectively . The undou women . A studyP< 0.05) , 37. The therapy , 39. How therapy . HoweverIt’s worth noting that we observed a complex non-linear association between LDL-C and diabetes as well as FBG in observational analysis, but no evidence of a causal nonlinear association in the MR analysis. This means more cautious interpretation needs to be considered in observational studies, especially when the prevalent non-communicable diseases globally have an evident close co-morbid trend. Shared risk factors may induce more confounding factors and heterogeneity may induce information bias that masks true associations. It is a good attempt to supplement more accurate biological phenotypes as secondary outcomes in observational studies to reveal the intricate mechanisms. It appeals for an integrated and prospective interrogation into their intercorrelation. In this study, we observed the reverse effects of LDL-C against FBG in observational and MR analyses. This may be attributed to the heterogeneity of diabetes. Klimentidis et al. found that levels of circulating LDL-C were negatively associated with type 2 diabetes prevalence , despite positive associations of circulating LDL-C with HbA1c and BMI . They spOur study is subject to several limitations. First, we used prevalent diabetes instead of incident diabetes in the FISSIC study. Although MR analyses were used to circumvent potential confounding and reverse causation, the results from observational studies are not as direct as that from prospective studies, which limits inferences and directions related to the causality. As incident diabetes cases develop in further research, it will be important to examine the association of LDL-C at baseline with incident diabetes. Second, we did not cover other specific diabetes-related phenotypes, such as HbA1c, a long-term glycaemic control indicator that helps distinguish patients with poor long-term glycaemic control from those with stress hyperglycemia, or HOMA-IR, the most popular indicator to qualify the degree of insulin resistance. In addition, although the participants in our study were aged ≥40 years, which avoided the confounding of type 1 diabetes to some extent, we could not further refine diabetes into more precise subtypes, but only generalized the cases to type 2 diabetes. And there were not sufficient data for the other complications. This limited our further exploration of underlying mechanisms by which LDL-C impacts the risk of diabetes, especially when diabetes is a heterogeneous disease overtly. Finally, our MR analysis was underpowered for the association of LDL with FBG and diabetes but was adequately powered for the association of LDL with diabetes among female subjects who did not receive the lipid-lowering drug.In conclusion, we verified the liner protective effect of LDL-C against diabetes among the female subjects who did not receive lipid-lowering drugs. Non-linear associations between LDL-C against diabetes in observational analysis are not causal, and prospective cohort study is welcomed to address this issue and to further explore the underlying mechanisms.https://shiny.cnsgenomics.com/mRnd/). Nonlinear MR available in the nlmr R package (https://github.com/jrs95/nlmr).We estimated statistic power for the MR studies using the online website . The patients/participants provided their written informed consent to participate in this study.DC conceived the study, undertook project leadership. In addition, DC, TW, and YW were guarantors of this work. YM wrote the first draft of the manuscript, analyzed data and interpreted the results. ZZ, XL, KD, and HX were involved in the data collection. All authors contributed to the drafting and critical revision of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China .We thank the research participants and researchers for their help in data collection and methodology.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nonobvious early symptoms are a prominent characteristic of pancreatic cancer, resulting in only 20% of patients having resectable tumors at the time of diagnosis. The optimal management of unresectable advanced pancreatic cancer (UAPC) remains an open research question. In this study, the tumors shrank significantly after PD-1 antibody combined with chemotherapy in two UAPC patients, and both have achieved R0  resection and survival to date.Case 1: A 53-year-old man was diagnosed with pancreatic adenocarcinoma (Stage III). He received six cycles of PD-1 antibody plus chemotherapy as the first-line treatment. The tumor was reduced from 11.8×8.8 cm to “0”  after preoperative neoadjuvant therapy (PNT) and the adverse effects were tolerable. The patient underwent radical surgery and achieved R0 resection. Case 2: A 43-year-old man diagnosed with pancreatic adenocarcinoma with liver metastasis (Stage IV) received three cycles of PD-1 antibody combined with chemotherapy. The tumor was reduced from 5.2×3.9 cm to 2.4×2.3 cm with no side effects. The patient also underwent radical surgery and achieved R0 resection.PD-1 antibody plus a chemotherapy regimen resulted in a surprising curative effect and safety in two patients with UAPC, which may portend an improvement in pancreatic carcinoma treatment. We may have a way for UAPC patients to obtain radical treatment and gain long-term survival. Two PD-L1 positive UAPC patients with microsatellite stability (MSS) enlighten us to have a more comprehensive understanding of the prediction of immunotherapy. Pancreatic cancer is a digestive system malignant tumor with an increasing incidence rate and a high mortality. In recent years, it has become an increasingly common cause of cancer-related death. Patients with different degrees of pancreatic cancer are divided into resectable, borderline resectable, and unresectable  pancreatic carcinoma . Poor pr2. Laboratory findings were within normal limits, except the leukocyte level was 9.88×109/L , the creatinine was 43 µmol/L , and the carbohydrate antigen 125 (CA-125) level was 36.3 IU/ml . Upper abdomen enhanced computed tomography (ECT) demonstrated a low-density shadow 11.8×8.8 cm behind the head and body of the pancreas and a slightly low-density shadow 3.1×2.5 cm was observed in the left lateral lobe of the liver showing a “fast-in and fast-out” strengthening pattern. The portal vein was invaded and thinned by the shadow behind the head of the pancreas. Multiple enlarged lymph nodes were found in the porta hepatis and retroperitoneum; the diameter of larger one was about 4.0 cm. Biopsy revealed poorly differentiated pancreatic adenocarcinoma and no obvious cancer tissue in the liver  was 29.6 kg/m®) (Day 1), and 125 mg/m2 albumin-bound paclitaxel (Day 1 and Day 8) plus 1000 mg/m2 gemcitabine (Day 1 and Day 8) (AG) as the first-line treatment for each 21-day cycle. Low-frequency ultrasound cavitation sensitization therapy based on gemcitabine was added in the fourth cycle, and the tumor perfusion increased by 35% after the fourth treatment. At the end of six courses of treatment, the pancreatic head and body were normal on ECT, and a band-like slightly low-density shadow 1.7×0.7 cm was seen in the left lateral lobe of the liver. Several small lymph nodes were found near the head of the pancreas and retroperitoneum, and the larger one was about 0.7 cm  for each 21-day cycle, and his general condition was good with no clinical evidence of recurrence. The patient received a change to his chemotherapy drug regimen once the total AG levels had reached the upper limit.UAPC patient preoperative downstaging therapy was first, followed by surgery. On November 21, 2020, the patient began receiving a PD-1 inhibitor, 200 mg Tislelizumab  level was 474 IU/ml  and the carbohydrate antigen 50 (CA-50) level was 354 IU/ml . ECT showed a round low-density mass of 5.2×3.9 cm in the tail of the pancreas surrounding the splenic artery and this had an unclear boundary with the coeliac trunk. The mass had invaded the splenic vein and lesser curvature of the stomach. Multiple round low-density shadows were seen in the right anterior lobe of the liver; the larger one was about 0.7×0.8 cm. Finally, the patient was diagnosed with both pancreatic adenocarcinoma and metastatic liver adenocarcinoma upon biopsy (2 irinotecan (Day 1) for each 21-day cycle. There was no clinical evidence of recurrence in the 12 months after resection and follow-up treatment continued.PD-1 antibody and AG were considered first as a downstaging therapy for the metastatic pancreatic cancer patient. Ultrasound-guided radiofrequency ablation of liver metastases was performed with Child-Pugh grade A score during the first treatment cycle. The low-density lesions in the liver became inactive high-density lesions after ablation. At the end of three rounds of treatment, ECT showed a sheet-like low-density mass 2.4×2.3 cm in the tail of the pancreas. Multiple round low-density shadows were seen in the liver, and the larger one was about 1.8×1.3 cm  has a good curative effect and relatively little toxicity and is a commonly used chemotherapy for pancreatic cancer. However, the rich stroma in pancreatic cancer makes it difficult for chemotherapy drugs to penetrate, resulting in few patients achieving a partial response through chemotherapy alone . RecentlPD-1 antibody plus chemotherapy could attain better therapeutic effects, which have been seen in other tumors such as gastric cancer and lung cancer , 12. TheTislelizumab is a humanized recombinant immunoglobulin G4 monoclonal antibody against PD-1, which blocks the PD-1/PD-L1 signaling pathway and releases the “brakes” of the immune system. It is currently the only PD-1 monoclonal antibody successfully modified on the Fc, which minimizes binding to the Fcγ receptor (FcγR) on the surface of macrophages and avoids the consumption of T cells caused by antibody-dependent cell-mediated phagocytosis (ADCP) . TisleliPredictive markers are critical for improving treatment outcomes. PD-1 antibodies have received FDA approval for treating deficiency mismatch repair/high microsatellite instability (dMMR/MSI-H) or non-high tumor mutation burden (TMB-H) tumors. However, the clinical benefit of immune checkpoint inhibitors in treating pancreatic cancer remains poorly understood and is unable to effectively predict those patients most likely to respond to ICI treatment. The two cases in this report both had MSS, but showed extremely high sensitivity to PD-1 antibody Table 1.Our study contributes to the general knowledge base about how best to treat patients with UAPC. Two patients herein underwent PNT (PD-1 antibody immunotherapy combined with chemotherapy) and ultimately achieved R0 resection. However, there were limitations such as a lack of control cases and a short observation period, which could not show postoperative improvement in overall survival. In the future, larger samples in clinical trial are needed to verify the clinical efficacy of our neoadjuvant treatment model in UAPC.The original contributions presented in the study are included in the article supplementary material. Further inquiries can be directed to the corresponding author.Written informed consent was obtained from all patients for the publication of any potentially identifiable images or data included in this article.LS and PL provided patient information. JF collected and analyzed the data. CZ and QB took care of the patients. XN took pathology photos. ZY and YK participated in treatment plans. TZ, BX, and JD helped with revising the manuscript. LS, PL, and HX wrote the paper. All authors contributed to the article and approved the submitted version.We thank Professor Yue Shuqiang of the Department of Hepatobiliary and Pancreatic Surgery, Xijing Hospital, Air Force Medical University, for providing surgical guidance. We thank the patients for allowing us to share their medical history and clinical course.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Importantly, in an on-body setup, the device achieved a significant amperometric response to sweat glucose in a very short amount of time (a few seconds). With detailed investigations, this proof-of-concept study could help further the development of sensitive and selective sweat-based glucose sensing devices for real-time glucose monitoring in diabetes patients.Wearable sweat sensors offer the possibility of continuous real-time health monitoring of an individual at a low cost without invasion. A variety of sweat glucose sensors have been developed thus far to help diabetes patients frequently monitor blood glucose levels through sweat glucose as a surrogate marker. The present study demonstrates the development and characterization of a three-dimensional paper-based microfluidic electrochemical integrated device (3D PMED) for measuring glucose concentration in sweat in real-time via simple, non-invasive, capillary-action-based sample collection. The device was selective for glucose, and it detected glucose accurately in the clinically relevant range (0~2 mM) in an off-body setup. To the best of our knowledge, this is the first time NEXAR™ has been used for biosensing applications. Further, the developed glucose sensor has acceptable sensitivity of 16.8 µA/mM/cm Wearable microfluidic sensors form a rapidly evolving field of research in biomedical engineering. They offer several advantages such as low cost, the ability to track an individual’s health status continuously (or semi-continuously) in real-time, non-invasive  testing of analytes and health parameters, and at-home and on-body testing ,2.Sweat sensors are among the most commonly developed and studied wearable sensors ,4,5,6,7.2 to 230 µA/mM/cm2 [A variety of natural sweat glucose sensors have been studied thus far to help diabetes patients frequently monitor blood glucose levels through sweat glucose as a surrogate marker ,10,11,12A/mM/cm2 ,14,15. SHowever, sweat sensors are associated with numerous unaddressed challenges. These include issues with sweat collection, the impact of external environmental factors on sweat production rate, and handling battery requirements for continuous electrolyte monitoring . Particu® membrane to improve the electrolyte separation capacity of the biosensor and thus reduce interference and improve selectivity. Both off-body and on-body tests of the fabricated device were performed in this proof-of-concept study.Our research demonstrates the development and characterization of a highly selective 3D PMED paper-based microfluidic electrochemical integrated device for measuring glucose concentration in natural sweat in real-time via simple, non-invasive, capillary-action-based sample collection. Importantly, NEXAR™  was used in place of the more commonly used Nafionw/v) and acetic acid (3% v/v) were purchased from Aladdin Ltd.  Sodium chloride, glucose , and NEXAR™ were obtained from Sigma-Aldrich.The following reagents were used: glucose oxidase  was obtained from Sigma-Aldrich . Phosphate-buffered saline  was obtained from Sangon Biotech  Co., Ltd., and chitosan  purchased from PalmSens B.V. . The SPCEs used in the present study had a conventional three-electrode configuration, with carbon as the working electrode (WE), silver as the reference electrode (RE), and carbon as the counter electrode (auxiliary electrode). Additionally, a screen mesh, wax (from crayons), and Whatman filter paper (cellulose paper) were used. Amperometric measurements were obtained using the PalmSens3™ potentiostat , controlled by PSTrace software version number 5.8, PalmSens B.V. . All measurements were carried out at room temperature unless specified otherwise.v/v) were drop-casted on the working electrode’s surface. NEXAR™ helps separate interfering electroactive agents in sweat. The coating was allowed to air-dry for 30 min at constant room temperature. Thereafter, 3 µL of a chitosan solution (5% w/v in 3% v/v acetic acid) were cast on the electrode and subsequently left overnight (8 h) at 4 °C. Subsequently, 3 µL of GOx (10 mg/mL) were drop-casted on the surface of the working electrode, and the electrode was allowed to dry for 2 h at room temperature. Note that GOx acts as the catalyst for the conversion of glucose to gluconolactone, releasing hydrogen peroxide (H2O2) in the process. Electrochemical reduction results in the subsequent amperometric response recorded in this study. In the fourth step, 3 µL of a chitosan solution (5% w/v in 3% v/v acetic acid) were dropped on the electrode and allowed to dry at room temperature. Lastly, 2 µL of the Nexar™-ethanol mixture (1:10 v/v) were dropped on the working electrode, allowed to dry, and then stored at 4 °C until further use , silver as the reference electrode (RE), and carbon as the counter electrode (auxiliary electrode) . This thther use .Whatman qualitative cellulose filter paper was glued to the back of the screen mesh to prevent the paper from sliding during the process of screen printing. The screen mesh was rubbed and consequently heated (at 50–55 °C). Distinct patterns, as shown in The glucose sensor (electrode) was tested in an off-body setup by adding increasing concentrations of glucose  onto the electrode surface. A calibration curve was generated based on the recorded amperometric response of the electrode. Thereafter, the integrated paper-based microfluidic was tested by adding glucose onto the collector end of the 3D PMED in 0.5 mM increments at intervals of 20 s. This experiment was used to measure the sensitivity of the device.Finally, on-body testing of the paper-based microfluidic was performed. Briefly, a study participant was enrolled after the necessary information regarding the research study was provided to them. This was carried out in accordance with the “Moroccan Foundation for Advanced Science, Innovation and Research (MAScIR)” Ethical Committee. For the study, the paper-based microfluidic was securely placed on the forearm of the study participant, who was then asked to engage in physical exercise (cycling). The amperometric response of the device based on real-time sampling of sweat glucose was recorded and subsequently plotted.The FESEM images of the screen-printed carbon electrodes at different preparation stages are displayed in Our developed glucose sensor was first used in an off-body setup to confirm its ability to detect glucose in simulated sweat (0.01 M PBS solution). A significant, reproducible, and sustained amperometric response was detected upon the successive addition of 0.1 mM glucose (in 0.01 M PBS) onto the electrode surface .2 = 1), as shown in With stepwise increases in glucose concentration, the current response also increased, as can be seen in 2 (approximate electrode surface area), which equals 16.8 µA/mM/cm2.After testing the glucose sensor independently, it was integrated with the 3D PMED. Subsequently, glucose solution was added to the collector of the 3D PMED in 0.5 mM increments . The highest amperometric differential (0.42 µA) was noted upon the first addition of 0.5 mM glucose . Thus, tMoreover, the resulting amperometric response was found to vary linearly with time . The resIn order to confirm that the amperometric current response noted in After confirming its functionality off-body, the 3D PMED was used for on-body glucose measurements in sweat. The device was placed on the study participant’s forearm , one of The present work was a proof-of-concept study wherein a 3D PMED was developed to determine the glucose concentration in sweat, and its application was demonstrated on a human participant.The 3D PMED glucose sensor developed in the present study was found to be selective for glucose. Note that sweat is primarily composed of water (99%), while the remaining 1% of the sweat is a mixture of ions , micronutrients , amino acids (such as histidine), glucose, urea, ammonia, lactate, pyruvate, and other metabolites (including drug metabolites) ,17,18. I®-based devices have also shown similarly high selectivity towards glucose [The primary reason for the glucose selectivity observed in the present study was the use of the NEXAR™ coating. Some previous studies on sweat metabolite analysis used a Nafion membrane instead of NEXAR™ to separate critical interfering electroactive molecules in sweat (such as chloride ions) from the absorbed sweat samples ,20. Nafi glucose ,21. It s glucose . This is®, making it more suitable for sweat-based biosensing applications. As a result of its greater water uptake capacity, NEXAR™ also has a significantly higher conductivity (2.63–4.84 mS/cm) than Nafion® (0.645–0.703 mS/cm) [NEXAR™ was previously used for desalination, oil separation, water filtration, and antimicrobial applications ,23. Howe3 mS/cm) .2, and the device was able to measure glucose in the 0–2 mM range, compatible with the range observed for sweat glucose. Future investigations should also include the evaluation of the device’s limit of detection by successively adding increasing amounts of glucose and registering the amperometric response yielded by the device. The smallest amount that is able to achieve a discernible amperometric response will be the limit of detection.It is important to note that glucose concentration in sweat , degradation of chemicals between collection and analysis, inability or difficulty to collect the required amount of sweat sample (preferably greater than 10 µL), and the need for expensive, voluminous instrumentation . Wearabl2/h), while at a higher intensity exercise regimen , the forehead is the site of the highest sweat rate (1710 g/m2/h) [One of the limitations of the present study is the use of a single study participant. To extend the results of this study beyond its current proof-of-concept stage, it is necessary to include a greater and statistically relevant number of study participants. It is also important to test the developed 3D PMED under different exercise regimens and in human participants with different skin conditions. Moreover, the sweat collection site of the 3D PMED should also be altered to test if eccrine gland sites other than the forearm  are better suited for sweat glucose quantitation. This is because the sweat rate for an individual depends not only on the intensity of the exercise regimen but also on the location of the body ,27. In a2.The present study demonstrates the development and characterization of a 3D paper-based microfluidic electrochemical integrated device (3D-PMED) for measuring glucose concentration in sweat in real-time via simple, non-invasive, capillary-action-based sample collection. The device was selective for glucose and was able to detect glucose in the clinically relevant range of 0–2 mM in an off-body setup. Further, the developed glucose sensor had an acceptable sensitivity of 16.8 µA/mM/cmOn-body testing has been performed in many different ways in previously reported studies. For example, in one study, the sensor was sewn onto an arm guard, and the study participants were asked to run for 25 min before on-body sweat glucose levels were recorded . Another